HQNO

Pseudomonas aeruginosaandStaphylococcus aureusare two of the most common coinfecting bacteria in human infections, including the cystic fibrosis (CF) lung. There is emerging evidence that coinfection with these microbes enhances disease severity and antimicrobial tolerance through direct interactions. However, one of the challenges to studying microbial interactions relevant to human infection is the lack of experimental models with the versatility to investigate complex interaction dynamics while maintaining biological relevance. Here, we developed a model based on anin vitromedium that mimics human CF lung secretions (synthetic CF sputum medium [SCFM2]) and allows time-resolved assessment of fitness and community spatial structure at the micrometer scale. Our results reveal thatP. aeruginosaandS. aureuscoexist as spatially structured communities in SCFM2 under static growth conditions, withS. aureusenriched at a distance of 3.5 μm fromP. aeruginosaMultispecies aggregates were rare, and aggregate (biofilm) sizes resembled those in human CF sputum. Elimination ofP. aeruginosa's ability to produce the antistaphylococcal small molecule HQNO (2-heptyl-4-hydroxyquinolineN-oxide) had no effect on bacterial fitness but altered the spatial structure of the community by increasing the distance ofS. aureusfromP. aeruginosato 7.6 μm. Lastly, we show that coculture withP. aeruginosasensitizesS. aureusto killing by the antibiotic tobramycin compared to monoculture growth despite HQNO enhancing tolerance during coculture. Our findings reveal that SCFM2 is a powerful model for studyingP. aeruginosaandS. aureusand that HQNO altersS. aureusbiogeography and antibiotic susceptibility without affecting fitness.IMPORTANCEMany human infections result from the action of multispecies bacterial communities. Within these communities, bacteria have been proposed to directly interact via physical and chemical means, resulting in increased disease and antimicrobial tolerance. One of the challenges to studying multispecies infections is the lack of robust, infection-relevant model systems with the ability to study these interactions through time with micrometer-scale precision. Here, we developed a versatilein vitromodel for studying the interactions betweenPseudomonas aeruginosaandStaphylococcus aureus, two bacteria that commonly coexist in human infections. Using this model along with high-resolution, single-cell microscopy, we showed thatP. aeruginosaandS. aureusform communities that are spatially structured at the micrometer scale, controlled in part by the production of an antimicrobial byP. aeruginosaIn addition, we provide evidence that this antimicrobial enhancesS. aureustolerance to an aminoglycoside antibiotic only during coculture.

Cystic fibrosis (CF) patients chronically infected with bothPseudomonas aeruginosaandStaphylococcus aureushave worse health outcomes than patients who are monoinfected with eitherP. aeruginosaorS. aureusWe showed previously that mucoid strains ofP. aeruginosacan coexist withS. aureusin vitrodue to the transcriptional downregulation of several toxic exoproducts typically produced byP. aeruginosa, including siderophores, rhamnolipids, and HQNO (2-heptyl-4-hydroxyquinolineN-oxide). Here, we demonstrate that exogenous alginate protectsS. aureusfromP. aeruginosain both planktonic and biofilm coculture models under a variety of nutritional conditions.S. aureusprotection in the presence of exogenous alginate is due to the transcriptional downregulation ofpvdA, a gene required for the production of the iron-scavenging siderophore pyoverdine as well as the downregulation of the PQS (Pseudomonasquinolone signal) (2-heptyl-3,4-dihydroxyquinoline) quorum sensing system. The impact of exogenous alginate is independent of endogenous alginate production. We further demonstrate that coculture of mucoidP. aeruginosawith nonmucoidP. aeruginosastrains can mitigate the killing ofS. aureusby the nonmucoid strain ofP. aeruginosa, indicating that the mechanism that we describe here may functionin vivoin the context of mixed infections. Finally, we investigated a panel of mucoid clinical isolates that retain the ability to killS. aureusat late time points and show that each strain has a unique expression profile, indicating that mucoid isolates can overcome theS. aureus-protective effects of mucoidy in a strain-specific manner.IMPORTANCECF patients are chronically infected by polymicrobial communities. The two dominant bacterial pathogens that infect the lungs of CF patients areP. aeruginosaandS. aureus, with ~30% of patients coinfected by both species. Such coinfected individuals have worse outcomes than monoinfected patients, and both species persist within the same physical space. A variety of host and environmental factors have been demonstrated to promoteP. aeruginosa-S. aureuscoexistence, despite evidence thatP. aeruginosakillsS. aureuswhen these organisms are coculturedin vitroThus, a better understanding ofP. aeruginosa-S. aureusinteractions, particularly mechanisms by which these microorganisms are able to coexist in proximal physical space, will lead to better-informed treatments for chronic polymicrobial infections.

Glycosylation of natural and synthetic products can alter the physical, chemical and pharmacological properties of the aglycon. Conversion of 2-heptyl-1-hydroxyquinolin-4-one (HQNO), a potent respiratory inhibitor produced by Pseudomonas aeruginosa, to the less toxic 2-heptyl-1-(β-D-glucopyranosydyl)-quinolin-4-one, was recently demonstrated for Bacillus subtilis strain 168. In this study, we compared the genomes of several Bacillus spp. to identify candidate enzymes for HQNO glucosylation. All three (putative) UDP-glycosyltransferases (GT) of B. subtilis 168 tested, YjiC, YdhE and YojK, were capable of HQNO glucosylation, with YjiC showing the highest turnover rate (kcat) of 4.6 s-1, and YdhE exhibiting the lowest Kmvalue for HQNO of 9.1 μM. All three GT predominantly utilized UDP-glucose, but YdhE was similarly active with TDP-glucose. Among the aglycons tested, HQNO was the preferred substrate of all three GT, but they also showed activities toward the P. aeruginosa exoproducts pyocyanin, 2-heptyl-3-hydroxyquinolin-4(1H)-one (the Pseudomonas quinolone signal) and 2,4-dihydroxyquinoline, the plant derived antimicrobials vanillin and quercetin, and the macrolide antibiotic tylosin A. Our results underline the promiscuity and substrate flexibility of YjiC, YdhE and YojK, and suggest a physiological role in natural toxin resistance of B. subtilis. Especially YdhE appears to be an attractive biocatalyst for the glycoengineering of natural products.

The opportunistic pathogen Pseudomonas aeruginosa, one of the most prevalent species in infections of the cystic fibrosis lung, produces a range of secondary metabolites, among them the respiratory toxin 2-heptyl-1-hydroxyquinolin-4(1H)-one (2-heptyl-4-hydroxyquinoline N-oxide, HQNO). Cultures of the emerging cystic fibrosis pathogen Mycobacteroides abscessus detoxify HQNO by methylating the N-hydroxy moiety. In this study, the class I methyltransferase MAB_2834c and its orthologue from Mycobacterium tuberculosis, Rv0560c, were identified as HQNO O-methyltransferases. The P. aeruginosa exoproducts 4-hydroxyquinolin-2(1H)-one (DHQ), 2-heptylquinolin-4(1H)-one (HHQ), and 2-heptyl-3-hydroxyquinolin-4(1H)-one (the 'Pseudomonas quinolone signal', PQS), some structurally related (iso)quinolones, and the flavonol quercetin were also methylated; however, HQNO was by far the preferred substrate. Both enzymes converted a benzimidazole[1,2-a]pyridine-4-carbonitrile-based compound, representing the scaffold of antimycobacterial substances, to an N-methylated derivative. We suggest that these promiscuous methyltransferases, newly termed as heterocyclic toxin methyltransferases (Htm), are involved in cellular response to chemical stress and possibly contribute to resistance of mycobacteria toward antimicrobial natural compounds as well as drugs. Thus, synthetic antimycobacterial agents may be designed to be unamenable to methyl transfer. ENZYMES: S-adenosyl-l-methionine:2-heptyl-1-hydroxyquinolin-4(1H)-one O-methyl-transferase, EC 2.1.1.